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RFPS Technology Our proprietary core technologies focus on protease research field. Our mission is the research, development and commercialization of products and services of the highest possible quality for scientists working in the protease field. Characterization of a protease involves identification of target
peptide sequences, measurement of activities toward these sequences
and determination of kinetic parameters. Commercially available substrates
are chemical substrates carrying chemical molecules fused to peptide
sequences. These chemical modifications often change the approach
of an enzyme toward the substrate providing biased or wrong information
on protease characteristics. Med Discovery's RFPS system (Felber et al. 2004. Biotechniques 36(5):878-85) allows the use of recombinant protein substrates presenting the protease substrate sequence in a fully biological surrounding, thus eliminating the risk of non-specific interactions with artificially introduced chemical compounds. The utilization of fluorescent proteins to monitor protease activity guarantees for an extremely high sensitivity of the system. Applications Characterization of substrate in a protein context Technical Background RFPS substrates consist of two fluorescent proteins which are covalently connected by a short peptide linker. This custom defined linker peptide harbours the protease substrate sequence. Proteolytic cleavage of this linker can be measured spectroscopically to determine kinetics of substrate hydrolysis by the protease. The RFPS system is based on fluorescence resonance energy transfer (FRET) between the donor and acceptor protein fluorophores. The emission spectrum of the donor protein overlaps the excitation spectrum of the acceptor enabling a transfer of energy. FRET only occurs if the two fluorophores are in very near vicinity of each other. Cleavage of the substrate by a protease separates the two fluorophores and results in a loss of the energy transfer (A). Thus, hydrolysis of the substrate can be evaluated by the measurement of increasing fluorescence intensity of the donor (Em. 485nm) and simultaneously decreasing fluorescence of the acceptor (Em. 528nm). Resistance to cleavage maintains the vicinity of fluorescent proteins and stable fluorescence at 485 and 528 nm (B).
High versatility of the RFPS system Use of various buffer systems: phosphate, citrate, acetate, Tris
HCl A detailed Protocol is included in each Data Sheet provided with every RFPS product. |
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